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R E S E A R C H A R T I C L E Open Access
N-acetyl-cysteine exhibits potent
anti-mycobacterial activity in addition
to its known anti-oxidative functions
Eduardo P. Amaral
1,2
, Elisabete L. Conceição
3,4
, Diego L. Costa
1
, Michael S. Rocha
3
, Jamocyr M. Marinho
5,6
,
Marcelo Cordeiro-Santos
7,8
, Maria Regina D’Império-Lima
2
, Theolis Barbosa
3,4
, Alan Sher
1
and Bruno B. Andrade
1,3,9,10*
Abstract
Background: Mycobacterium tuberculosis infection is thought to induce oxidative stress. N-acetyl-cysteine (NAC)
is widely used in patients with chronic pulmonary diseases including tuberculosis due to its mucolytic and
anti-oxidant activities. Here, we tested whether NAC exerts a direct antibiotic activity against mycobacteria.
Methods: Oxidative stress status in plasma was compared between pulmonary TB (PTB) patients and those
with latent M. tuberculosis infection (LTBI) or healthy uninfected individuals. Lipid peroxidation, DNA oxidation
and cell death, as well as accumulation of reactive oxygen species (ROS) were measured in cultures of primary human
monocyte-derived macrophages infected with M. tuberculosis and treated or not with NAC. M. tuberculosis,
M. avium and M. bovis BCG cultures were also exposed to different doses of NAC with or without medium
pH adjustment to control for acidity. The anti-mycobacterial effect of NAC was assessed in M. tuberculosis
infected human THP-1 cells and bone marrow-derived macrophages from mice lacking a fully functional
NADPH oxidase system. The capacity of NAC to control M. tuberculosis infection was further tested in vivo
in a mouse (C57BL/6) model.
Results: PTB patients exhibited elevated levels of oxidation products and a reduction of anti-oxidants compared
with LTBI cases or uninfected controls. NAC treatment in M. tuberculosis-infected human macrophages resulted
in a decrease of oxidative stress and cell death evoked by mycobacteria. Importantly, we observed a dose-dependent
reduction in metabolic activity and in vitro growth of NAC treated M. tuberculosis,M. avium and M. bovis
BCG. Furthermore, anti-mycobacterial activity in infected macrophageswasshowntobeindependentof
the effects of NAC on the host NADPH oxidase system in vitro. Short-term NAC treatment of M. tuberculosis
infected mice in vivo resulted in a significant reduction of mycobacterial loads in the lungs.
Conclusions: NAC exhibits potent anti-mycobacterial effects and may limit M. tuberculosis infection and
disease both through suppression of the host oxidative response and through direct antimicrobial activity.
Keywords: Tuberculosis, N-acetyl cysteine, Antimicrobial activity, Therapy
* Correspondence: bruno.andrade@bahia.fiocruz.br
1
Immunobiology Section, Laboratory of Parasitic Diseases, National Institute
of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD
20892, USA
3
Laboratório Integrado de Microbiologia e Imunorregulação (LIMI), Instituto
Gonçalo Moniz, Fundação Oswaldo Cruz (FIOCRUZ), Salvador 40296-710,
Bahia, Brazil
Full list of author information is available at the end of the article
© The Author(s). 2016 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Amaral et al. BMC Microbiology (2016) 16:251
DOI 10.1186/s12866-016-0872-7
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Background
N-acetyl-cysteine (NAC) is included in the World Health
Organization’s list of essential medicines; a list that details
the most relevant medications needed for a basic health
system [1]. Acetyl-cysteine is a derivative of cysteine in
which an acetyl group is attached to nitrogen. Due to its
disulfide reducing activity, NAC is used as a mucolytic
agent to promote expectoration [2]. NAC is commonly
prescribed as an adjunct therapy in patients with a wide
range of respiratory diseases characterized by formation
of thick mucus, such as cystic fibrosis [2–4]. At high
doses, NAC results in significantly improved small airway
function and decreased exacerbation frequency in patients
with stable chronic obstructive pulmonary disease (COPD)
[3, 4]. NAC’s mucolytic activity is also the basis of its
use in liquefying sputum samples for the microscopic
detection of acid-fast bacilli (AFB) in suspected pulmonary
tuberculosis (TB) patients [5]. Furthermore, in both ex-
perimental animal models and clinical studies, NAC dis-
plays a protective effect on acute liver injury induced by
anti-TB drugs in acetaminophen-dependent or independ-
ent conditions [6–11]. In patients with type 2 diabetes,
NAC holds promise in primary prevention of cardiovas-
cular complications and systemic inflammation [12–14].
In addition to the above clinical applications, NAC
has been employed as a potent anti-oxidant in several
experimental models of infection and cancer in vitro
and in vivo [15–20]. In these settings, NAC serves as a
pro-drug to L-cysteine, which is a precursor to the bio-
logic antioxidant glutathione. This anti-oxidant property
of NAC is associated with strong anti-inflammatory
effects, which have been suggested to inhibit the activation
of nuclear factor-κB(NF-κB) with subsequent inhibition
of cytokine synthesis [2, 21, 22]. In a mammalian model
of Mycobacterium tuberculosis infection, NAC has been
shown to diminish TB-driven lung pathology and inflam-
matory status, as well as to reduce mycobacterial infection
loads in the lung [23]. These effects were attributed to the
drug’s anti-oxidant properties and immune regulatory
activities. Whether NAC limits M. tuberculosis infection
in this situation through a direct microbicidal effect on M.
tuberculosis was not addressed. Indeed, NAC has been
shown to exhibit anti-microbial activity against a number
of bacterial pathogens including Pseudomonas aeruginosa,
Staphylococcus aureus, Helicobacter pylori, Klebsiella
pneumoniae and Enterobacter cloacae [17, 24–26].
In this study, we demonstrate that NAC directly impairs
the growth of several species of mycobacteria in vitro
independent of its inhibitory effects on the host NADPH
oxidase system. This anti-mycobacterial effect was also
observed in an experimental model in vivo. Thus, NAC
may limit M. tuberculosis infection and disease both
through suppression of the host oxidative response and
through direct antimicrobial activity. This dual host and
pathogen directed function makes the drug an interesting
candidate for use as adjunct therapy for tuberculosis.
Methods
Clinical study
Cryopreserved heparinized plasma samples were collected
from 30 subjects with active pulmonary TB (20 males;
median age 32 years, interquartile range [IQR]: 18–47), 20
individuals with LTBI (10 males; median age 31 years,
IQR: 22–46) and 20 healthy controls (8 males; median age
20 years, IQR: 19–38). The study groups were similar with
regard to age (p = 0.248) and gender (p = 0.162). Subjects
were recruited between May and November 2012 at the
Hospital Especializado Octávio Mangabeira, Salvador,
Brazil, as part of a biomarker study [27]. Tuberculosis
diagnosis included positive AFB in sputum smears and
positive M. tuberculosis sputum cultures. Three sputum
samples per subject were examined by fluorescence
microscopy, processed by the modified Petroff’smethod
and cultured on Lowenstein-Jensen medium. LTBI diag-
nosis was performed in contacts of active TB cases who
agreed to participate in the study. Diagnosis was based on
tuberculin skin test (TST) positivity (≥10 mm in diam-
eter), absence of chest radiography abnormalities or pul-
monary symptoms and negative sputum cultures. Healthy
control subjects (health care professionals and medical
students from the Hospital Especializado Octávio Manga-
beira who agreed to participate) were asymptomatic with
normal chest radiograph and negative sputum cultures
and TST induration (<5 mm in diameter). At the time of
enrollment, all individuals were HIV negative (all patients
were actively screened), BCG-vaccinated and had no
record of prior TB disease or of anti-TB treatment.
Mice
C57BL/6 mice, male, 6–8 weeks old (n= 20), were
purchased from Taconic Farms (Hudson, NY). The
gp91Phox
−/−
mice, male, 6–8 weeks old (n=10), geneti-
cally backcrossed in the C57BL/6 background were a kind
gift from Dr. Sharon Jackson (NIMHD, NIH). These mice
do not exhibit a fully functional NADPH oxidase system
[28, 29]. All mice were maintained in micro-isolator cages
under controlled temperature and humidity. They were
fed ad libitum at NIAID animal facilities.
Bacteria
The H37Rv M. tuberculosis (ATCC),Beijing 1471 hyper-
virulent M. tuberculosis [30], Mycobacterium avium
SmT 2151 and Mycobacterium bovis BCG, strains were
used. Mycobacteria from a single colony-forming unit
(CFU) were suspended in Middlebrook 7H9 medium
(Difco, BD Biosciences, USA) that was supplemented
with 10 % albumin-dextrose-catalase (ADC; Difco) and
0.05 % Tween 80 (Sigma-Aldrich, USA), cultivated and
Amaral et al. BMC Microbiology (2016) 16:251 Page 2 of 10
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then frozen at −80 °C in aliquots of 10
8
bacilli/mL. Prior
to performing the experiments, the aliquots were thawed
and diluted in complete 7H9 medium. To avoid bacterial
clumps, the samples were sonicated for 30 s and homog-
enized. The bacilli were quantified by spectrophotometry
at 600 nm. In some experiments, the pH in Middlebrook
7H9 medium was adjusted in acidic conditions (pH 5.8)
and neutral conditions (pH 6.8–7.4) as indicated in
the figures.
Cell cultures
CD14
+
column-purified human elutriated monocytes
were obtained from peripheral blood of healthy donors
at the NIH blood bank. Macrophages were generated by
culturing monocytes in the presence of RPMI media
containing 10 % human AB serum and M-CSF 50 ηg/
mL (Prepotech, Rocky Hill, NJ) for 7 days, with fresh
media with growth factor being added every 48 h as
previously described [31]. For infection assays, cells were
plated at the concentration of 10
6
cells/well in 24-well
plates in phenol and serum free media (Opti-MEM; Life
Technologies, Carlsbad, CA).
The human monocyte-like cell line (ATCC) THP-1
was differentiated into mature macrophages by treatment
with phorbol ester (PMA; Sigma Aldrich, USA). Briefly,
THP-1 cells were cultured in RPMI medium (Gibco;
1 mM sodium pyruvate, 2 mM glutamine) supplemented
with 10 % fetal calf serum at 37 °C in 5 % CO
2
. For the
experiments, THP-1 cells were seeded on 96-well plate at
10
5
cells/well in the presence of PMA (40 ηmol/L) for
24 h to induce macrophage differentiation [32, 33]. Cells
were washed and incubated for additional 24 h in fresh
medium without PMA until their use in experiments.
After infection with mycobacteria, the macrophage cultures
were washed to remove extracellular bacteria and then
cells were cultured in phenol and serum free media
(Opti-MEM).
To obtain mouse bone marrow derived macrophages
(BMDM), bone marrow cells were cultivated in 30 % L929
cell-conditioned medium for 7 days as previously de-
scribed [34]. An additional 10 ml of L929 cell-conditioned
medium were added after 4 days of incubation. BMDMs
were detached with cold PBS and seeded in 96-well plates
at 10
5
cells/well, containing serum and phenol free media
at 37 °C in 5 % CO
2
.
In experiments testing the effects of NAC on myco-
bacterial infection in vitro, cells (BMDM or THP-1
macrophages) were infected with H37Rv, M. avium or
M. bovis BCG at MOI of 10 for 3 h, washed and then
cultivated for 5 days. Bacterial uptake was evaluated
at different time points by measuring CFU in the infected
BMDM cultures following treatment with 0.05 % saponin
(Sigma-Aldrich, USA) for 10 min [30].
Mycobacterial quantification
Bacterial numbers were determined by serial dilution of
bacterial cultures and cell lysates in Middlebrook 7H11
medium (Difco) supplemented with 10 % oleic acid/
albumin/dextrose/catalase (OADC; Difco). CFU numbers
were determined after 3 weeks of incubation at 37 °C
in 5 % CO
2
.
Determination of mycobacterial metabolic activity
Metabolic activity of bacilli was evaluated using a tetra-
zole salt assay in a 96-well plate as described previously
[35]. Briefly, bacteria were pipetted in a 96-well plate at
10
6
CFU/well (50 μL), and NAC (50 μL) diluted in
Middlebrook 7H9 added at the indicated concentrations.
Plates were then incubated at 37 °C in 5 % CO
2
for 5 days
of indicated time points. After this period, tetrazolium
salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazole)
at 5 mg/mL (final concentration) was added to the cul-
tures for 3 h. Next, 100 μL of lysis buffer (20 % w/v SDS/
50% DMF –dimethylformamide in distilled water) was
added and the cultures were incubated overnight at 37 °C
in 5 % CO
2
. Bacterial cultures treated with rifampin
(1 μg/mL; Sigma-Aldrich) were used as a positive control
for growth inhibition. Middlebrook 7H9 alone was used
as a negative control. The samples were read using a
spectrophotometer at 570 nm.
Chromatographic and immunological assays
Total oxidant status was assessed using an enzymatic
assay kit from Rel Assay Diagnostics (Gaziantep, Turkey)
following the manufacturer’s protocol. The results are
expressed in terms of micro molar hydrogen peroxide
equivalent per liter (μmol H
2
O
2
Equiv./L). Total antio-
xidant status was measured using the Antioxidant Assay
kit from Cayman Chemical (Ann Harbor, MI). In this
assay, the capacity of the antioxidants to prevent ABTS
(2,2′-azino-di-[3-ethylbenzthiazoline sulphonate]) oxida-
tion is compared with that of Trolox, a water-soluble
tocopherol analogue, and is quantified as molar Trolox
equivalents. Lipid peroxidation in plasma and culture
supernatants was quantified using an assay kit from
Cayman Chemical, which measures the formation of mal-
ondialdehyde (MDA). DNA/RNA oxidation was measured
in culture supernatants using an immunoassay from
Cayman Chemical that detects all three oxidized guanine
species: 8-hydroxy-2′-deoxyguanosine from DNA, 8-hy
droxyguanosine from RNA, and 8-hydroxyguanine from
either DNA or RNA. Results from this assay were shown
as concentration of 8-OH-DG. Cell death was estimated
by quantification of lactate dehydrogenase (LDH) in
culture supernatants using a kit from Cayman Chemical
following the manufacturer’s protocol. Cell death was
plotted as percentage of cell death compared with positive
control (H
2
O
2
). Intracellular production of ROS in primary
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human macrophages was assessed by staining cells with the
oxidative fluorescent dye probe, dihydroethidium (DHE)
5 mM (Invitrogen/Molecular Probes, Grand Island, NY)
for 30 min at 37 °C in 5 % CO
2
and then analyzed using a
flow cytometer. The cells used for the ROS measurement
assay were previously detached from the culture plates
using trypsin 0.25 %, washed and resuspended in phenol
and serum free medium. Results were plotted as histo-
grams where the mean fluorescence intensity (MFI) was
compared between the experimental groups.
In vivo experiments
Mice were anesthetized using ketamine (Vetbrands,
Brazil; 100 mg/kg) and xylazine (Vetbrands; 15 mg/kg),
diluted in sterile saline and administrated intraperito-
neally. Mice were infected with H37Rv M. tuberculosis
strain (approx. 3 × 10
4
bacilli) inoculated intratracheally
as described previously [30]. Mice were given NAC
(400 mg/kg) by gavage daily for 6 days. The dose of
NAC used was based on studies reported previously,
which describe the effect of NAC treatment during
chronic Mtb infection in vivo, as well as in other experi-
mental models [23, 36–38]. On day 7 of infection, mice
were euthanized using CO
2
exposure chamber, followed
by cervical dislocation, before lungs were harvested. For
CFU counting, lungs were homogenized in 1 mL of cold
PBS using a cell strainer (100 μm; Corning, USA) and
plated in Middelbrook 7H11 medium (Difco) agar plate.
Statistical analysis
The median values with interquartile ranges were used
as measures of central tendency. For in vitro experi-
ments, bars represent mean and standard errors. The
Mann-Whitney test (for two groups) or Kruskal-Wallis
with Dunn’s multiple comparisons or linear trend
post-hoc tests (for more than two groups) were used to
compare continuous variables. Differences observed in the
experimental studies were assessed using Student’st-test
(for comparisons between two groups) or One-Way
ANOVA with Tuckey post-test. A p-value of <0.05 was
considered statistically significant.
Results and discussion
NAC inhibits oxidative stress, lipid peroxidation, DNA
oxidation and cell death in M. tuberculosis-infected
human macrophages
Previous studies in active TB patients point to an asso-
ciation of this disease with excessive oxidative stress
by demonstrating decreased systemic concentrations of
antioxidants and enhanced spontaneous generation of free
radicals compared to individuals without TB [27, 39]. The
measurement of total oxidant status, total antioxidant
status and lipid peroxidation offers a reliable way to verify
that there is an imbalance between the production of free
radicals and the ability of the body to detoxify their effects
through neutralization by antioxidants. This imbalance of
oxidative stress status results in irreversible cell damage,
leading to many pathophysiological conditions [40–44].
To characterize the oxidative stress status during TB
infection we measured total oxidant status, total antio-
xidant status and lipid peroxidation in the plasma of
pulmonary TB (PTB) patients and those with latent
M. tuberculosis infection (LTBI) or healthy uninfected
individuals. Lipid peroxidation, DNA oxidation and cell
death, as well as accumulation of reactive oxygen species
(ROS) were also assessed in cultures of primary human
monocyte-derived macrophages infected with M. tubercu-
losis and treated or not with NAC.
Interestingly, PTB patients exhibited substantial eleva-
tion of total oxidation status (Fig. 1a) and lipid pero-
xidation (Fig. 1b) in the plasma while simultaneously
displaying a significant reduction in soluble antioxidants
(Fig. 1c) compared to LTBI cases or uninfected controls
(Fig. 1a-c). Following M. tuberculosis infection in vitro,
human monocyte-derived macrophages displayed aug-
mented lipid peroxidation (Fig. 1d), DNA oxidation
(Fig. 1e) and cell death induction (Fig. 1f), which were
accompanied by a dramatic accumulation of intracellular
ROS (Fig. 1g). In our experiments we found that treat-
ment of M. tuberculosis-infected macrophage cultures
with NAC (10 mM) significantly decreased ROS accu-
mulation, lipid peroxidation and DNA oxidation, while
restoring cell viability (Fig. 1d-g). Together these findings
support the concepts that TB is associated with excessive
oxidative stress and death in infected macrophages and
show that this response can be successfully reduced by
treatment with NAC.
NAC limits mycobacterial extracellular growth
independent of pH
A growing body of evidence suggests that NAC can
affect bacterial viability inside of eukaryotic host cells
[45]. To test this concept with mycobacteria we infected
THP-1 cells (to reduce variability between blood donors)
with M. tuberculosis,M. avium or M. bovis BCG bacilli
(MOI of 10) for 3 h, replaced the media, and added
NAC (10 mM) to half of the cultures. Intracellular
bacterial growth was then assessed 5 days later by lysing
the cells and measuring CFU. We observed a marked
reduction in bacterial loads for each of the mycobacterial
species in the NAC-treated cultures demonstrating the
ability of the drug to limit mycobacterial growth within
THP-1 cells (Fig. 2a). Although minor bacterial growth
occurred in presence of NAC it was clearly inhibited when
it was compared to control (untreated cells).
High concentrations of NAC have been shown to
directly inhibit extracellular growth of a number of bacte-
rial species [24, 25, 46]. Indeed, NAC inhibits biofilm
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formation of a variety of medically important gram-posi-
tive and gram-negative bacteria, e.g. P. aeruginosa, K.
pneumoniae, S. epidermidis, S. aureus and Escherichia
coli [25, 26, 46–48]. Interestingly, NAC does not inhibit
growth of methicillin-sensitive and resistant S. aureus
strains at 49 mM (8 mg/mL) [49], suggesting that bacterial
species may diverge in susceptibility to the effects of this
drug. Here, we tested whether NAC could affect the
growth of different strains of mycobacteria in cell free cul-
ture broth. Bacterial metabolic activity and CFU numbers
were reduced in a dose dependent manner at day 5 follo-
wing treatment with NAC (Fig. 2b and c). Moreover, a
hypervirulent M. tuberculosis Beijing strain (Beijing 1471)
[30, 50] was also highly susceptible to NAC treatment
(Additional file 1: Figure S1). Next, we performed a kinetic
experiment in which M. tuberculosis was cultured in the
presence or absence of NAC in a wide range of concentra-
tions for different days and bacterial growth was assessed
by CFU counts. We observed a striking >2log
10
reduction
in CFU counts in cultures treated with NAC 10 mM
Fig. 1 NAC reverts M. tuberculosis-induced oxidative stress. Cryopreserved heparinized plasma samples collected from active pulmonary
TB (PTB; n= 30), latent TB individuals (LTBI; n= 20) and healthy controls (HC; n= 20) from Salvador Brazil were used in these studies.
Total oxidant status (a), lipid peroxidation (b) and total antioxidant status (c) were measured as described in Methods. d-f Primary
human monocyte-derived macrophages were infected with H37Rv M. tuberculosis at MOI of 5 and treated or not with NAC (10 mM).
Lipid peroxidation (d), DNA oxidation (e) and cell death (f) were assessed at indicated time points post-infection (p.i.) as described in
Methods. gIntracellular production of ROS in primary human macrophages infected with H37Rv M. tuberculosis at different MOI was
measured by flow cytometry. ROS production was also verified in infected-macrophages after NAC treatment (10 mM). Results were
plotted as histograms where the mean fluorescence intensity (MFI) was compared between the experimental groups. In a-c lines represent
median values. In d-g data represent means ± SEM of triplicate samples from a minimum of six donors. In a-f data were analyzed using
Kruskal-Wallis test with Dunn’s multiple comparisons post-test. In gMOI titration data were compared using Kruskal-Wallis test with
non-parametric linear trend post-test. The effect of NAC treatment was analyzed using the Mann-Whitney Utest. The data shown are
representative of three independent experiments. (*p<0.05; **p<0.01, ***p< 0.001)
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(Fig. 3a) at 5 days and mycobacterial sterilization in the
cultures after 7 days of treatment. These findings demon-
strate that NAC exhibits a potent anti-mycobacterial effect
while dampening infection-induced oxidative stress, pro-
moting increased survival of infected cells.
In our experimental conditions, we observed that the
pH of cellular or mycobacterial growth media dropped
in the presence of NAC (from 6.8 to 5.8) at 10 mM. We
tested whether the doses of NAC used to demonstrate
inhibition of mycobacterial growth in vitro maybe toxic
for human cells. Uninfected human macrophage-like
THP-1 cells were cultured for 24 h in the presence of
different concentrations of NAC in media with or
without pH adjustment (pH = 7.4 in adjusted conditions
and pH = 5.8 in unadjusted conditions). Cell viability was
assessed by flow cytometry (using frequency of cells
staining positive for UV live/dead). We observed that
NAC did not affect THP-1 cell viability at 10 mM,
but exhibited significant pH dependent cytotoxicity at
higher concentrations such as 100 mM (Additional file 2:
Figure S2A).
A low pH of medium can potentially limit the growth
of some bacteria in vitro [51]. However, mycobacterial
species have been reported to grow under acidic condi-
tions in complex media [52]. To test whether the modifi-
cation of cell culture pH by addition of NAC could
explain the changes in mycobacterial growth observed
here, we cultivated mycobacterial strains in media with
either acidic pH (pH = 5.8) or neutral pH (pH ~7.4).
Importantly, the pH adjustment of the treated mycobac-
terial cultures only partially reduced the capacity of NAC
to inhibit the metabolic activity of the bacilli and the
bacteria growth (Additional file 2: Figure S2B and C). Of
note, acidification of bacterial medium, to a pH similar to
Fig. 2 NAC restrains mycobacterial growth within THP-1 macrophages and exhibits a direct anti-mycobacterial effect on extracellular bacteria
in vitro. aHuman-THP-1 macrophages were infected with M. tuberculosis,M. avium or M. bovis strains at an MOI of 10 for 3 h. Extracellular
bacteria were removed by washing. Cells were then cultivated for 5 days in the presence of NAC at 10 mM. CFU counts were assessed as
described in Methods. band cMycobacteria strains were grown in Middlebrook 7H9 supplemented with OADC in 96-well plates. Metabolic
activity measurements (b) and CFU counts (c) were performed as described in Methods. Significant differences were observed for the
indicated experimental conditions compared to untreated cultures (*p< 0.05; **p< 0.01, ***p< 0.001). The data represent the means ± SEM
of triplicate samples. The data shown are representative of three independent experiments
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what is observed during NAC treatment, did not
affect M. tuberculosis growth(Fig.3b).Theseresults
argue that NAC has direct antimicrobial activity that
compromises mycobacterial growth independent of
acidification of culture pH.
NAC promotes reduction of bacterial burden in infected
mice
We further examined whether NAC exhibits anti-myco-
bacterial activity in vivo. C57BL/6 mice were intratra-
cheally infected with M. tuberculosis (~3 × 10
4
forms) and
then treated with NAC (400 mg/kg daily) via gavage start-
ing on day 1 of infection, and continuing for 6 days
(Fig. 4a). Bacterial burden was measured on day 7 post-
infection to evaluate any potential early bactericidal activ-
ity (EBA) in vivo. Interestingly, short-term NAC treatment
of mice infected with H37Rv M. tuberculosis in vivo re-
sulted in a significant reduction of mycobacterial loads in
the lungs compared to those of untreated animals (Fig. 4b),
suggesting that NAC is able to limit mycobacterial growth
in vivo. In agreement with these findings, a recent study
revealed that oral treatment of M. tuberculosis-infected
guinea pigs with NAC for up to 60 days post-infection re-
duces both bacterial burden and extra-pulmonary disease
severity [23]. Nevertheless, the effects of NAC in that study
were attributed solely to its antioxidant capacity whereas
we observed an additional direct anti-mycobacterial effect
of NAC treatment. Whether the same dual effects of NAC
treatment would be observed in NAC treated TB patient
remains to be investigated.
The antimicrobial activity of NAC against mycobacteria is
not dependent on the host NADPH oxidase system
NAC is a known antioxidant and is commonly used as a
ROS scavenger in diverse scenarios [2]. NADPH oxidase
is a major source of intracellular ROS in eukaryotic cells
[28, 29]. To determine whether NAC acts independently
of NADPH-derived ROS inside macrophages we infected
gp91Phox
−/−
deficient mouse macrophages, which lack
the main NADPH subunit and thus lack NADPH acti-
vity [29], with M. tuberculosis in the presence or absence
of non-host cell cytotoxic doses of the compound
(10 mM). We observed that treatment with NAC inhibited
bacteria proliferation in both wild type and gp91Phox
−/−
macrophages (Fig. 5a and b, respectively), indicating that
the antimicrobial activity of NAC is not dependent on a
fully functional host NADPH oxidase system.
Fig. 3 NAC limits mycobacterial proliferation by acting as an
anti-mycobacterial compound. Mycobacterium tuberculosis was
grown in Middlebrook 7H9 supplemented with OADC as
described in Methods. aKinetic of mycobacteria growth during
7 days in the presence of NAC at indicated concentrations was
verified using CFU counts. bMycobacterial growth was evaluated
at different pH as described in Methods. Fold increase of bacterial
growth was calculated as the ratio of CFU number counted on
days 0 and 5. Significant differences were observed for the indicated
experimental conditions (***p<0.001). The data represent the means
± SEM of triplicate samples. The data shown are representative of at
least three independent experiments
Fig. 4 Short-term NAC treatment results in a significant reduction
of mycobacterial loads in the lungs from mice infected with
M. tuberculosis.aC57BL/6 mice were intratracheally infected
with~3×10
4
bacilliofH37Rvstrain.Miceweretreatedornot
with NAC (400 mg/kg) by gavage daily for 6 days. bOn day 7
p.i., animals were euthanized and lungs were harvested. CFU/g
values in lungs were determined as described in Methods. Data
represent individual values and means ± SEM from a total of 5
animals per group. Data from two independent experiments are
shown. Significant differences were observed for the indicated
groups (**p< 0.01)
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NAC is a precursor of both the amino acid L-cysteine
and of reduced glutathione (GSH) [2]. In a previous
study of HIV-infected patients, in vitro NAC treatment
led to increased levels of GSH inside macrophages and
subsequent infection of these macrophages with M.
tuberculosis resulted in reduction of intracellular bacterial
counts [53]. Once produced, GSH has been described to
limit bacterial growth directly through a mechanism
dependent on the mycobacterial enzyme gamma-glutamyl
transpeptidase [54]. This enzyme cleaves GSH and S-
nitrosoglutathione to form the dipeptide cysteinylglycine
(Cys-Gly), which is transported to the interior of the
bacterial cell by the multicomponent ABC transporter
dipeptide permease [54, 55]. In addition, a mutant M.
tuberculosis strain lacking expression of gamma-glutamyl
transpeptidase has been shown to grow normally in the
presence of GSH [54]. Therefore, one possibility on how
NAC is involved in anti-mycobacterial effects of patho-
genic mycobacteria is that NAC may exert a bactericidal
effect on mycobacteria through the generation of GSH
and in turn Cys-Gly. Additional studies using mycobac-
teria deficient in gamma-glutamyl transpeptidase could be
used to test this hypothesis.
Conclusions
The observation that NAC exerts anti-mycobacterial
activity in vivo has previously been interpreted as resul-
ting solely from the anti-oxidant properties of the com-
pound. In light of our discovery of a direct antimicrobial
function of NAC against different mycobacterial strains,
this conclusion should be reassessed. Our finding that
NAC possesses dual modes of action suggests that NAC
warrants re-examination as a therapeutic treatment of
mycobacterial infection. Shorter anti-TB treatment is
needed in view of the high rate of drug toxicity, cost and
complexity of the current 6-month daily regimen [56].
Accordingly, clinical trials of this potential adjunct anti-
TB therapy would provide a good prospect for reducing
the current long-term treatment against TB.
Additional files
Additional file 1: Figure S1. NAC inhibits the growth of hypervirulent
Beijing strain in vitro.Beijing 1471 M. tuberculosis strain was grown in
Middlebrook 7H9 supplemented with OADC with or without NAC
(10 mM). CFU counts were performed as described in Methods.
Significant differences were observed for the indicated experimental
conditions compared to untreated cultures. The data represent the
means ± SEM from two independent experiments. (**p< 0.01). (TIF 48 kb)
Additional file 2: Figure S2. NAC inhibits mycobacterial growth in
vitro independent of pH. (A) Uninfected THP-1 cells were incubated
with different concentrations of NAC at varying pH (acidic pH (pH 5.8)
or neutral pH (pH ~7.4)). Cellular viability was assessed using live/dead
staining and analyzed by flow cytometry (B and C). Mycobacteria
strains grown in Middlebrook 7H9 supplemented with OADC were
exposed to different concentrations of NAC at the indicated pH.
Metabolic activity measurement (B) and CFU counts (C) were performed
as described in Methods. Significant differences were observed for the
indicated experimental conditions compared to untreated cultures
(**p< 0.01, ***p< 0.001). The data represent the means ± SEM of triplicate
samples. The data shown are representative of at least two independent
experiments. (TIF 1382 kb)
Abbreviations
ABTS: 2,2′-azino-di-[3-ethylbenzthiazoline sulphonate]; AFB: Acid-fast bacilli;
BMDM: Bone marrow derived macrophages; CFU: Colony-forming units;
COPD: Chronic obstructive pulmonary disease; Cys-Gly: Cysteinylglycine;
DHE: Dihydroethidium; EBA: Early bactericidal activity; GSH: Glutathione;
IQR: Interquartile range; LDH: Lactate dehydrogenase; LTBI: Latent M.
tuberculosis infection; MDA: Malondialdehyde; MFI: Mean fluorescence
intensity; NAC: N-acetyl-cysteine; NF-κB: Nuclear factor-κB; PTB: Pulmonary TB;
ROS: Reactive oxygen species; TB: Tuberculosis; TST: Tuberculin skin test
Acknowledgments
We are grateful to Melissa Schechter (Sackler School of Medicine, Tel Aviv
University, Israel) for critical review of the manuscript. We also thank Deborah
Surman (NIAID, NIH) and Sandra D. Oland (NIAID, NIH) for excellent technical
support.
Funding
This work was supported by the Intramural Research Program of the
National Institute of Allergy and Infectious Diseases, National Institutes
of Health and by the Fundação de Amparo à Pesquisa do Estado de
São Paulo-FAPESP grant 2010/51150-4, 2013/07298-5 and 2015/19126-0.
The funders had no role in study design, data collection and analysis,
decision to publish, or preparation of the manuscript.
Authors’contributions
Conceived and designed the experiments: EPA BBA. Performed the
experiments: EPA BBA DLC. Analyzed the data: EPA BBA. Contributed
reagents/materials/analysis tools: EPA ELC MSR JMM MCS MRDL TB AS
BBA. Wrote the paper: EPA BBA. All authors read and approved the final
manuscript.
Competing interests
The authors declare that they have no competing interests.
Ethics approval and consent to participate
All clinical investigations were conducted according to the principles
expressed in the Declaration of Helsinki. Written informed consent was
obtained from all participants or their legally responsible guardians before
enrolling into the study. The clinical study was approved by the Ethical
Committee of the Centro de Pesquisas Gonçalo Moniz, Fundação
Fig. 5 Anti-mycobacterial property of NAC occurs independently
of the host NADPH oxidase system. BMDMs generated from
aC57BL/6 and bgp91Phox
−/−
mice were infected with M. tuberculosis
and intracellular growth of the bacteria was assessed as described in
Methods. The data represent the means ± SEM of triplicate samples.
Significant differences were observed for the indicated groups
(*p< 0.05). The data shown are representative of at least two
independent experiments
Amaral et al. BMC Microbiology (2016) 16:251 Page 8 of 10
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Oswaldo Cruz (FIOCRUZ) (protocol number: 003.0.225.000-11).
All animal studies were approved by the National Institutes of Health
(NIH) Institutional Animal Care and Use Committee (IACUC) and the
experiments were carried out in accordance with the approved guidelines.
Author details
1
Immunobiology Section, Laboratory of Parasitic Diseases, National Institute
of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD
20892, USA.
2
Department of Immunology, Laboratory of Immunology of
Infectious Diseases, Institute of Biomedical Science, University of São Paulo,
São Paulo 05508-900, Brazil.
3
Laboratório Integrado de Microbiologia e
Imunorregulação (LIMI), Instituto Gonçalo Moniz, Fundação Oswaldo Cruz
(FIOCRUZ), Salvador 40296-710, Bahia, Brazil.
4
Instituto de Ciências da Saúde
(ICS), Universidade Federal da Bahia, Salvador 40110-100, Brazil.
5
Departament of Internal Medicine, School of Medicine and Public Health,
Salvador 41150-100, Brazil.
6
Programa de Controle da Tuberculose, Hospital
Especializado Octávio Mangabeira, Salvador 40320-350, Brazil.
7
Departamento
de Ensino e Pós-Graduação, Fundação de Medicina Tropical Dr. Heitor Vieira
Dourado, Manaus, Brazil.
8
Programa de Pós-Graduação em Medicina Tropical,
Universidade do Estado do Amazonas, Manaus, Brazil.
9
Multinational
Organization Network Sponsoring Translational and Epidemiological
Research (MONSTER) Initiative, Fundação José Silveira, Salvador 45204-040,
Brazil.
10
Curso de Medicina, Faculdade de Tecnologia e Ciências, Salvador
41741-590, Brazil.
Received: 23 February 2016 Accepted: 26 October 2016
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